Chlorella virus-encoded endonucleases are a major new resource of fine structure gene mapping tools for molecular biology. In contrast to bacterial restriction enzymes, which recognize 4 to 8 base pair sequences, Cvi endonucleases cut at 2 to 4 b.p. sites. By suitably optimizing conditions of virus infection and enzyme extraction conditions, it is possible to produce commercial-scale quantities of Cvi enzymes from virus-infected cells. Phase II research is proposed to further develop the Chlorella virus system for production of over 100,000 units of several novel 2 to 4 b.p. Cvi endonucleases. Furthermore, a large-scale screening program for new Cvi enzymes is proposed, based on a proprietary mass screening protocol. These new Cvi endonucleases will have widespread applications in high resolution physical mapping of chromosomes, medical diagnostics, DNA forensics, population genetics, and other fine structure gene mapping applications. PROPOSED COMMERCIAL APPLICATION: Chlorella virus endonucleases have a large market potential for basic and applied research in molecular biology, agriculture, medicine, and population genetics. Furthermore, since these enzymes function at ambient temperatures, they lend themselves to applications in field studies or remote locations. Cvi endonucleases have a very short product development time.